The molybdenum cofactor (Moco) is present in four different enzyme families which catalyze important transformations in the global carbon, sulfur and nitrogen cycles. Moco consists of a mononuclear molybdenum coordinated to the sulfur atoms of a dithiolene group which is part of a substituted pterin derivative termed molybdopterin. Enzymes containing this cofactor are found in all three phylogenetic kingdoms of life, and biosynthesis of the cofactor follows the same pathway, both in prokaryotes and in eukaryotes including plants and human. Moco deficiency is inherited as an autosomal recessive trait and is a severe disease in humans which manifests itself in neurological abnormalities leading to premature death in the affected individuals. The experiments outlined in this proposal address two important aspects of Moco, namely its biosynthesis and its detailed function once it has been incorporated into the target enzymes. The specific aims presented in this proposal therefore are: (i) Characterization of the early steps in Moco biosynthesis through structural studies on the enzyme MoaC. MoaC most likely either catalyzes the first step in Moco biosynthesis starting with a phosphorylated guanosine, or the second step in which A molybdopterin precursor is generated which still lacks the sulfur atoms. (ii) Crystal structure analysis of molybdopterin synthase which incorporates the sulfur atoms into a molybdopterin precursor generating molybdopterin. The small subunit of the heterodimeric molybdopterin synthase contains an unusual C-terminal thiocarboxylate which serves as the sulfur donor during catalysis. (iii) Crystal structure determination of MogA which might function as a molybdochelatase and thus would be responsible for incorporating molybdenum, which is present in the cell as molybdate, into the molybdopterin. (iv) High resolution structure analysis of the Moco-containing enzyme dimethylsulfoxide reductase including characterization of different oxidation states and complexes of the enzyme with substrates and inhibitors which will lead to a detailed characterization of the catalytic mechanism. (v) Crystal structure analysis of biotin sulfoxide reductase, which is related in sequence to dimethylsulfoxide reductase but differs in substrate specificity. Comparative studies of the two enzymes will lead to a more general understanding of the roles Moco is playing in enzymes containing this cofactor.